Basophil degranulation test

ABSTRACT

The present invention concerns a method for determining the degranulation activity of basophilic granulocytes in a sample as well as a reagent kit that is suitable for this. Furthermore the invention also concerns a method for diagnosing allergic hypersensitivity and for monitoring the response to a hyposensitization therapy.

[0001] The present invention concerns a method for determining thedegranulation activity of basophilic granulocytes in a sample as well asa reagent kit that is suitable for this. In addition the invention alsoconcerns a method for diagnosing allergic hypersensitivity and theresponse to a hyposensitization.

[0002] The classical classification of allergies according to Coombs andGell still largely applies nowadays as a basis for understanding theallergic type 1 reaction of the immediate type and is clearly defined byan IgE-mediated immune response. T cells with a particular cytokinepattern (interleukin 4 and interleukin 5) play a special role in this.As a result B lymphocytes are stimulated to switch to IgE productionwhich only occurs at very low levels in the serum and only develops itsfatal effect after cell binding to high affinity receptors andsubsequent cell degranulation. The cytokines, in particular interleukin5, additionally cause an increased maturation of promyelocytes intoeosinophilic granulocytes which cause delayed damage especially to thebronchial system by the release of toxic proteins from their granula.The physiological relevance of the type 1 allergy is still completelyunclear as well as the reason why the T cells secrete a cytokine patternfor an IgE response.

[0003] The determination of specific IgE antibodies in the serum (RAST,CAP etc.) is well established and is especially helpful when knownallergens need to be tested or when a skin test is dangerous ortechnically impossible for example in skin diseases or under theinfluence of drugs. In these methods the allergens are bound covalentlyor with high affinity to matrices with a large surface (sponges, beads,paper discs), subsequently incubated with patient serum and then, afterwashing, a detection is carried out using a radioactive orenzyme-labelled anti-human-IgE antibody. The amount of bound IgE isstated semiquantitatively in so-called RAST and CAP classes orquantitatively in kU IgE/l. The evaluation can be carried outradiometrically (RAST) or for example, fluorimetrically (CAP, enzymewith a fluorogenic substrate).

[0004] The advantages of these tests are that they are easy to carry outand there is a good correlation with the gold standard skin test foraeroallergens and so-called atopens. However, serious disadvantages mustbe accepted. These disadvantages are for example the lack of rareallergens and often epitope changes due to coupling of the allergens tothe matrix. In addition a carrier molecule such as human serum albuminis required for smaller allergens (haptens). Further difficulties arecaused by the fact that the specificities of serum IgE andmembrane-bound IgE on basophils and mast cells are not necessarily thesame since only a slow exchange takes place. Also the threshold ofdegranulation readiness (releasability) is not detected. Correlationwith the response to a hyposensitization in the form of an IgE decreaseis only fairly satisfactory for insect venom allergens i.e. in themajority of cases there is no explanation. Furthermore the specific IgEvalues must be put into perspective in the case of atopic patients witha high IgE level i.e. the total IgE level of the patient must be known.The severity of the clinical picture hardly correlates with the level ofthe specific IgE concentration.

[0005] Ready-made test strips have been developed as a simplifiedversion of this method on which the various allergens are applied. Theyare evaluated by comparing the blue colour that forms with a colourscale. Inhalation antigens which produce a reaction in the higher RASTclasses are readily detected. Otherwise there is little comparativedata. The test is primarily conceived for allergists in private practicewho do not have a large laboratory.

[0006] Due to the problems mentioned above in determining specific serumIgE, tests have been developed in which the basophilic granulocytes aredegranulated after stimulation with an allergen and subsequently thereleased components of the granula are determined in the supernatant.Such components are for example histamine (Immunotech Co. IBL),leukotrienes (CAST-ELISA, Biermann Co.) or tryptase (Pharmacia Co.).These assays are designed as enzyme immunoassays in a batch procedureand require a duration of two days due to overnight incubation steps. Afurther disadvantage with regard to the histamine determination is theacylation step for the released histamine as well as a frequentlyexcessively high histamine content of the allergen solutions due tobacterial contaminations. Furthermore for economic reasons it isnecessary to collect the supernatants of several patients which delaysthe assessment. The degranulation must, however, be carried out on freshwhole blood on the day of blood withdrawal.

[0007] Therefore attempts have already been made to detect thedegranulation cytometrically. In one approach the absorption ofbasophilic granula was determined with a Technicon H6000 measuringinstrument. In this case the measurement points were counted by hand onthe screen (Nilsson, Eur. J. Haematol, 45 suppl 53, 50-54, 1990).Alternatively the axial loss of light is measured after staining withtoluidine blue in an Ortho cytofluorograph (Nakagawa et al. (Allergy 36,39-47, 1981). A further approach is to carry out a flow-cytometric twocolour immunofluorescence method in which differently labelled anti-CD45and anti-IgE antibodies are used (Gane et al., Cytometry 19, 361-365,1995). This method is based on a relative increase of the expressiondensity of CD45 and a reduction of the IgE antibody expression indegranulation. However, a disadvantage of this method is that the changein the CD45 expression density is only small.

[0008] None of these cytometric methods has been introduced for routineuse due to the said disadvantages since they are tied to special, notgenerally available instruments or the signal differences are veryslight. Furthermore it is difficult to measure the basophils since theyare rare in the blood and are very difficult to distinguish from otherleucocytes on the basis of scattered light properties alone.

[0009] Hence the object that forms the basis of the present invention isto provide a new method for determining the degranulation activity ofbasophilic granulocytes which can be carried out simply and rapidly andcan be performed with conventional flow cytometers which are widely usedin hospitals and laboratory practices.

[0010] This object is achieved by a method for determining thedegranulation activity of basophilic granulocytes in a sample, wherein

[0011] a) the sample is mixed and incubated with a test substance,

[0012] b) the sample is mixed and incubated with a first bindingmolecule which can bind to cell-bound IgE antibodies or high affinityreceptors thereof, and with a second binding molecule which can bind toa surface molecule which appears de novo on the surface of basophilicgranulocytes during degranulation, in which the first binding moleculeand the second binding molecule carry different marker groups that canbe detected concurrently or can each bind to different marker groupsthat can be detected concurrently, and

[0013] c) the labels of the binding molecules are determined separatelyand the degranulation activity in the sample is determined qualitativelyor/and quantitatively on the basis of this determination.

[0014] In the method according to the invention the basophils arefirstly unequivocally determined via the marker using a first specificbinding molecule e.g. an antibody. This first binding molecule binds tocell-bound IgE antibodies or high affinity receptors thereof. Onlybasophils have a high surface density of IgE molecules which are boundto high affinity receptors (Fc_(ε)R1). IgE-specific or receptor-specificantibodies are preferably used which do not lead to a cross-linking ofthe IgE.

[0015] A further special feature of the method according to theinvention is that a second binding molecule is used which can bind to anantigen that only appears on the surface as a result of degranulation(de novo expression). An example of such an antigen is the preformedlysosomal protein gp55 which does not appear on the surface until thegranula membrane has fused with the cytoplasmic membrane duringdegranulation and can be detected there.

[0016] The term “test substance” in the present invention is understoodas any molecule which can cause an activation of basophilic granulocytesor degranulation. The test substance is usually an allergen such asplant pollens, an insect venom such as bee or wasp venom, a drug such aspenicillin or a latex allergen. In addition “test substance” also refersto other biological and synthetic substances which can cause such anactivation and can be used in the sense of a positive control. Anexample of this is the chemotactic peptide fMLP.

[0017] In one embodiment of the method according to the invention thesample is preincubated with a stimulant such as a cytokine whichamplifies the degranulation activity. The amount of cytokine ispreferably 0.1-100 ng per test mixture. This preincubation leads to asensitization of the basophilic granulocytes which reduces the thresholdfor degranulation readiness (releasability). The stimulant used ispreferably a lymphokine such as an interleukin or a growth factor suchas a colony stimulating factor (CSF). IL-3 or GM-CSF are preferred.Preincubation with the cytokine is usually carried out before step (a)of the method according to the invention i.e. before adding theallergen, but it can also be carried out simultaneously with theallergen incubation.

[0018] Incubation with an allergen as the test substance in the case ofa sample derived from an allergy patient leads to an activation of thebasophilic granulocytes and thus to a degranulation the extent of whichcorrelates with the extent of the allergic reaction. The first bindingmolecule serves to define the basophilic granulocytes and the secondbinding molecule serves to define that portion of the basophilicgranulocytes in which a degranulation has taken place. The relativeproportion of the degranulated granulocytes can thus be determined in asimple manner from the ratio between the cells that are positive for thefirst and second binding molecule and the cells which are only positivefor the first binding molecule.

[0019] The antigen recognized by the second binding molecule is usuallya protein or a glycoprotein which only appears on the surface as aresult of degranulation and thus allows an unequivocal differentiationbetween degranulated and non-degranulated granulocytes. The surfacemolecule is preferably gp55.

[0020] Any biological substances can be used as the first and secondbinding molecule provided they bind specifically either to IgEantibodies or to their high affinity receptors or to adegranulation-specific surface molecule. An antibody or an antibodyfragment is preferably used as a first or/and a second binding molecule.Monoclonal antibodies are preferred but polyclonal antibodies are alsosuitable. In particular for the first binding molecule, those antibodiesor antibody fragments are suitable which have a high specificity but lowcross-linking or degranulation properties (e.g. Fab fragments).Antibodies or antibody fragments against the Fc_(ε) receptor areparticularly suitable as the first binding molecule since the cells inonly a small number of the patients are inadequately recognized andactivated by anti-IgE.

[0021] Furthermore it is preferred in the method according to theinvention to terminate the degranulation after a defined incubationperiod with the allergen and the first and second binding molecule. Thiscan for example be achieved by fixing the leukocytes. Furthermore it ispreferable to lyse the erythrocytes after the incubation. Lysis of theerythrocytes and fixing of the leukocytes can also optionally be carriedout in one step. Hence a solution is added to the sample which forexample contains a high concentration of ammonium chloride and EDTA.Solutions which contain formaldehyde and/or ethylene glycol are alsosuitable. A preferred solution for carrying out lysis and fixation is acommercially available solution under the trade name FACS lysingsolution (Becton Dickinson). A wash step is optionally carried out afterthe fixation.

[0022] In addition to the determination of IgE and de novo expressedsurface molecules, the method according to the invention can alsocomprise the determination of one or several additional parameters. Thusthe sample can be additionally incubated with a third binding moleculewhich is capable of binding to the surface molecule CD45 and carries amarker group or is capable of binding to a marker group which can bedetermined separately in addition to the marker groups of the first andsecond binding molecule. The third binding molecule is preferably ananti-CD45 antibody or a fragment of such an antibody.

[0023] The additional determination of CD45 whose expression isincreased after activation of the basophilic granulocytes furtherincreases the accuracy of the determination.

[0024] A further parameter that can also be determined in the methodaccording to the invention is the light that is scattered sideways whichincreases during degranulation of basophils.

[0025] The binding molecules used in the method according to theinvention can carry a marker group or can bind to a marker group. If thebinding molecule carries a marker group i.e. is directly labelled, it ispreferably present as a covalent conjugate with the marker group. On theother hand the binding molecule can also be indirectly labelled i.e. itdoes not directly carry a detectable marker group but can bind to afurther substance which carries the marker group. Examples of indirectlylabelled binding molecules are hapten-coupled antibodies which can bindto an anti-hapten antibody carrying a marker group. The coupling ofmarker groups and haptens to biological binding molecules, in particularto antibodies or antibody fragments, is known to a person skilled in thefield of immunological test procedures and does not therefore requirefurther elucidation. Directly labelled binding molecules are preferredfor the method according to the invention.

[0026] Essentially all known markers in the field of diagnosticdetection methods are suitable as marker groups for the bindingmolecules, where the only prerequisite is that the individual markerscan be determined concurrently. Fluorescent markers are preferred whicheach have a different spectral emission spectrum e.g. fluorescein,phycoerythrin (PE) and peridinium-chlorophyll-A protein (Per CP) or aPE-cyanine 5 tandem conjugate (e.g. cychrome). The determination ispreferably carried out in a flow cytometer.

[0027] According to another preferred embodiment of the present method aDNA dye which can be determined in addition to the marker on theanti-IgE antibody and receptor is added and determined as a furtherparameter. The DNA dye serves to stain the cell nuclei and thus toexclude thrombocytes and unlysed erythrocytes. A fluorescent dye such as7-aminoactinomycin D or propidium iodide is preferred.7-aminoactinomycin D is particularly preferred.

[0028] The sample used in the method according to the invention is abody fluid which contains granulocytes. A particular advantage is thatthe method according to the invention can be used on whole blood withoutrequiring a cell isolation of basophilic granulocytes. Heparinized wholeblood is preferred as the sample and it is generally derived from ahuman donor although other samples from mammals such as rodents can alsobe used.

[0029] The method according to the invention is very simple and rapid.It can be carried out on any commercial flow cytometer which are presentin all medium and large hospitals and laboratories.

[0030] The method conditions can be varied depending on the test formatused. When using fluorescent markers and a flow cytometric detection ofthe markers, suitable conditions for step (a) i.e. incubation with thetest substance have turned out to be a time period of 5 to 60 min,preferably of 20 to 40 min at a temperature of 25 to 40° C., preferablyof 35 to 39° C. A 30 min incubation at about 37° C. gives good results.Step (b) is preferably carried out in the cold in order to avoidunspecific degranulation and suitable conditions have turned out to be atemperature of 0 to 10° C., preferably a temperature of 0 to 5° C. for atime period of 10 to 40 minutes, preferably of 5 to 30 minutes. If afixation is carried out after step (b), a further cooling of the sampleis not essential since the fixation prevents further degranulation. Step(c), i.e. the determination of the markers, is carried out following theincubations and optionally lysis and subsequent wash steps. In thisconnection the measurement sample is adequately stable for a period ofat least one hour, preferably about two hours or more.

[0031] As is apparent to a person skilled in the art, it is expedient tomeasure suitable controls in a determination by the inventive method inwhich a mixture without the test substance is suitable as the negativecontrol and a substance that activates the granulocytes such as fMLP issuitable as a positive control. Furthermore it is advantageous to alsomeasure a sample whose values are essentially in the normal range suchas a body fluid from a healthy normal test person.

[0032] A further subject matter of the present invention is a reagentkit for the determination of the degranulation activity of basophilicgranulocytes in a sample. The reagent kit contains a first bindingmolecule which can bind to cell-bound IgE antibodies or their highaffinity receptors and a second binding molecule which can bind to asurface molecule which appears de novo on the surface of basophilicgranulocytes during degranulation in which the first and the secondbinding molecule carry different marker groups that can be detectedconcurrently or each can bind to different marker groups that can bedetected concurrently. Optionally the reagent kit can contain a thirdbinding molecule as an additional detection reagent which can forexample bind to the CD45 surface molecule and carries a marker groupwhich can be detected in addition to the markers of the first and secondbinding molecule or can bind to such a marker group. The reagent kit canalso contain a DNA dye. Further optional components of the reagent kitare test substances i.e. various allergens or positive controlsubstances such as fMLP. In addition the reagent kit can also containstimulants such as cytokines or/and reagents for lysing erythrocytesor/and for fixing leukocytes.

[0033] Furthermore the kit can additionally contain a reagent for lysingerythrocytes and/or for fixing leukocytes as well as optionallyconventional buffers, auxiliary substances or additives.

[0034] A preferred reagent kit contains for example at least one testsubstance, an anti-gp55-FITC antibody, an anti IgE-PE antibody, a FACSlysing solution as well as optionally 7-aminoactinomycin D or/and ananti-CD45-PerCP antibody and a stimulant such as IL-3 or GM-CSF.

[0035] Yet a further subject matter of the invention is a method fordiagnosing the allergic hypersensitivity of a patient or the response toa hyposensitization which is characterized in that the degranulationactivity of basophilic granulocytes is determined in a sample from apatient by means of the method according to the invention using one orseveral allergens and a positive control as test substances, thedegranulation activity in a negative control sample is determined in anadditional test mixture and the presence or the absence of an allergichypersensitivity or the success or failure of a hyposensitizationtherapy is determined by comparing the values that are obtained.

[0036] An appropriate negative control sample is a sample from the samepatient to which no test substance has been added in step (a) of themethod according to the invention and preferably a stimulated sample ofa healthy normal test person is also assayed as a second negativecontrol sample.

[0037] An additional subject matter of the invention is the use ofanti-gp55 antibodies in a method for determining the degranulationactivity of basophilic granulocytes and the use of the method andreagent kit according to the invention for the diagnosis of allergichypersensitivity and for monitoring the response to a hyposensitizationtherapy.

[0038] The method according to the invention is further elucidated bythe following examples in conjunction with the attached FIGS. 1-6.

[0039]FIG. 1: shows the evaluation of a test according to the inventionfor a negative control,

[0040]FIG. 2: shows the evaluation of a test according to the inventionfor a positive control,

[0041] FIGS. 3-5: show a comparison of tests according to the inventionwith a histamine release test of the prior art and

[0042]FIG. 6: shows the evaluation of a test according to the inventionwith prestimulation with IL-3.

EXAMPLE 1

[0043] Peripheral blood (heparinised) of the patient is incubated withvarious concentrations of the suspected allergen at 37° C. In the caseof a corresponding sensitization this leads to a degranulation of thebasophilic granulocytes and to an activation which is primarilycharacterized by expression of the gp-55 antigen. In addition theexpression density of the IgE receptor decreases and the density of CD45on basophils increases. The side scattered light increases (loss ofabsorbing granula) and the number of basophils in the scattered lightgate for lymphocytes decreases. The detection is carried out by a threecolour immunofluorescence and flow cytometric analysis. The chemotacticpeptide fMLP serves as a positive control and a buffer solution (PBS) isused as the negative control. Antihistamine administration inhibits thetest.

[0044] 1. Material

[0045] 1.1 Reagents

[0046] Sheath liquid: FACS-flow (Becton Dickinson Co. order No. 342003)or another carrier liquid with a low intrinsic fluorescence.

[0047] FACS lysing solution, 10×concentrate (Becton Dickinson Co. orderNo. 349202).

[0048] Wash solution: PBS (phosphate buffered saline without calcium andmagnesium) as a ready-to-use solution or as salts (e.g. Biochrom,Berlin) or PHAGO-BURST wash solution (ORPEGEN Pharma Co., Heidelberg).

[0049] Chemotactic Peptide

[0050] fMLP (2000×) (ORPEGEN Co.) diluted in PBS.

[0051] For the calibration of quantitative measurements: Calibritecalibration particles, Becton Dickinson, order No. 349502.

[0052] 1.2 Monoclonal Antibodies

[0053] a) anti gp55-FITC, (ORPEGEN Pharma), order No. 1464 (prediluted1:4 in the ready-made combination)

[0054] b) anti IgE-PE (ORPEGEN Pharma), order No. 1465 (prediluted 1:40in the ready-made combination)

[0055] c) anti CD45 PerCP (Becton Dickinson) or CD45 cychrome(Pharmingen), order No.

[0056] 1.3 Allergens

[0057] ALK depot SQ (Scherax) mixture of 6 grasses+rye 100,000 U/ml,order No. 145a/90.90c

[0058] latex allergen (ALYOSTAL ST-IR) order No. 0903

[0059] ALK-prick SQ (Scherax) bee venom, order No. 222a/85a, 300 μg/ml

[0060] ALK-prick SQ (Scherax) wasp venom, order No. 223a/85a, 300 μg/ml

[0061] Allergopen (penicillin G, PPL and derivative MDM), order no.162a/81,

[0062] penicillin “Grünenthal” 1 mega, PZN-7803133

[0063] mite allergen from the Geer Labs company among others.

[0064] 2. Procedure

[0065] 2.1 Sample Preparation

[0066] The sample to be examined is divided into aliquots of 100 μl ineach case. In the case of high leukocyte numbers (WBC of 10-20,000 C/μl)it is possible to use a smaller sample volume e.g. 50 μl. Multiple testmixtures are not necessary.

[0067] 2.2 Preparation of the Allergen Dilutions

[0068] ALK-depot SQ (Scherax) mixture of 6 grasses+rye is used atconcentrations of 10,000, 1,000, 100 and 10 U/ml. ALK-prick SQ (Scherax)bee venom and ALK-prick SQ (Scherax) wasp venom are used atconcentrations of 10, 1, 0.1 and 0.01 μg/ml. Latex allergens, PPL(allergopen) and MDM (allergopen) are used in dilutions of 1:10, 1:100and 1:1,000. Penicillin Grünenthal is used at concentrations of 100, 10,1 and 0.1 μg/ml. PBS serves as a negative control and fMLP at aconcentration of 10⁻⁷ M (i.e. 1:2,000 PBS) is used as the positivecontrol. A sample from a healthy normal test person is also assayed as afurther control.

[0069] 2.3 Incubation with Allergen

[0070] 100 μl of each of the allergen dilutions is added to an aliquotof the sample, mixed and incubated for 30 minutes at 37° C. in a waterbath. Then the mixtures are placed on ice.

[0071] 2.4 Addition of Antibodies

[0072] The following antibody solutions are then added.

[0073] a) 20 μl anti gp55-FITC (prediluted)

[0074] b) 20 μl anti IgE-PE (prediluted)

[0075] c) 10 μl anti CD45-cychrome or 20 μl CD45-PerCP

[0076] After antibody addition it is well mixed (vortex) and incubatedfor 20 minutes on ice while protected from light.

[0077] 2.5 Lysis and Fixing

[0078] 2 ml FACS lysing solution (erythrocyte lysis and leukocytefixation) is then added, it is mixed (vortex) and incubated for 10minutes at room temperature while protected from light. Subsequently itis centrifuged for 5 min at 1300 rpm or 300 g and decanted.

[0079] 2.6 Washing the Cells

[0080] The cells are washed by adding 3 ml wash solution (PBS),centrifuging (5 min at 1300 rpm or 300 g) and decanting.

[0081] 2.7 Measurement

[0082] The sample is measured by flow cytometry for example on an FACSscan instrument. The stability of the measured sample is presumably ≧2hours on flaked ice in the dark.

[0083] The instrument settings (“BASO-SET” in the program CellQuest®)can be selected as follows. Expected Parameter Amplifier valueresolution FSC lin E00 (2.0) 256 channels SSC lin 360 256 channels FL1log, 4 decades 620 256 channels FL2 log, 4 decades 500 256 channels FL3log, 4 decades 620 256 channels trigger on 100 FSC compensation FL1-%FL2  0.9 FL2-% FL1  21 FL2-% FL3  0.2 FL3-% FL2  29

[0084] filter combinations (not variable in the FACScan): Parameterspectrum fluorochrome filter FL1 fluorescence 1 green FITC, TOBP530+/−15 nm fluorescence FL2 fluorescence 2 orange PE, PI BP585+/−22nm fluorescence FL3 fluorescence 3 red PerCP, red613, LP>650 nmfluorescence Cy5, PI

[0085] In the SSC/IgE dot plot, a region is set around the cells withlow and high side scattered light and strong IgE expression. These cellsare optionally displayed in the dot plot FL3 (CD45) versus FL2 (IgE) aspart of the validation and the x and y mean values of this populationare evaluated and the FL1 histogram (gp 55). The markers are set suchthat the evaluation region covers the region of the gp55 positivebasophils (the PBS test mixture serves as a negative control). The meanvalue FL1 is observed in all (also gp55-negative) cell populations. Ifpossible, at least 1000 cells of the subclass of interest (in this casebasophils) are recorded. In the positive test mixture a bimodaldistribution is formed in which the activated basophilic granulocytesrepresent an individual population. It is therefore possible to view thepercentage which is adequate for evaluating the test result.

[0086] 3. Evaluation

[0087] The evaluation of a negative control containing PBS is shown inFIG. 1. The evaluation of a positive control containing fMLP is shown inFIG. 2.

[0088] The following Table 1 shows typical values where all parametersshould be taken into consideration as a plausibility check. A comparisonbetween the negative control (PBS) and positive control (fMLP) showsthat the percentage of basophils remains essentially constant, the CD45expression density increases after activation and the IgE densitydecreases slightly. The percentage of gp55-positive basophilicgranulocytes and the mean gp55 value increases strongly afteractivation. assay % basos mean CD45 mean IgE % gp55 mean gp55 PBS 2.50134 1032 3.3  33 fMLP 2.35 349  981 30.6 369

[0089] A comparison of the diagrams for CD45 and gp55 for the positiveand negative control shows that a considerably higherallergen-specifically induced increase of expression occurs in the caseof gp55.

[0090] Results of the method according to the invention are shown inFIGS. 3-6. FIG. 3 shows a comparison of the method according to theinvention (gp55 or CD63 expression) with a conventional histaminerelease test for the determination of a grass pollen allergy.Corresponding data are shown in FIG. 4 for the determination of a beevenom allergy and in FIG. 5 for the determination of a wasp venomallergy. An example of a latex allergen determination shown in FIG. 6shows that the degranulation activity of basophils can be considerablyincreased by prestimulation with a cytokine like IL-3.

[0091] Legends

[0092] FSC=forward light scatter=narrow angle forward light scatter=cellsize

[0093] SSC=side scatter=orthogonal light scatter=cell granularity

[0094] algE-PE=IgE-PE=monoclonal antibody against immunoglobulin E,directly conjugated with the fluorescent dye phycoerythrin.

[0095] gp55-FITC=monoclonal antibody against the lysosomal glycoproteinwith a molecular weight of 55 kD, directly conjugated with thefluorescent dye fluorescein.

[0096] CD45 cychrome=monoclonal antibody against the common cell surfaceglycoprotein of leukocytes directly conjugated with the tandem dyePE-cyanin 5.

[0097] R1, R2, R3=regions in which certain cells are located and whichfunction as a logical gate. In this case they are the typical regionsfor basophilic granulocytes.

[0098] M1=marker region in which the cells which react positively to theantigen gp55 are located (degranulated basophils).

1. Method for the determination of the degranulation activity ofbasophilic granulocytes in a sample, wherein a) the sample is mixed andincubated with a test substance, b) the sample is mixed and incubatedwith a first binding molecule which can bind to cell-bound IgEantibodies or high affinity receptors thereof, and with a second bindingmolecule which can bind to a surface molecule which appears de novo onthe surface of basophilic granulocytes during degranulation, in whichthe first binding molecule and the second binding molecule carrydifferent marker groups that can be detected concurrently or can eachbind to different marker groups that can be detected concurrently, andc) the labels of the binding molecules are determined separately and thedegranulation activity in the sample is determined qualitatively or/andquantitatively on the basis of this determination.
 2. Method as claimedin claim 1, wherein the sample is preincubated with a stimulant thatamplifies the degranulation activity.
 3. Method as claimed in claim 2,wherein a cytokine, in particular IL-3 or GM-CSF is used as a stimulant.4. Method as claimed in one of the claims 1 to 3, wherein an antibody oran antibody fragment is used as the first binding molecule or/and as thesecond binding molecule.
 5. Method as claimed in one of the previousclaims, wherein an anti-IgE antibody or a fragment thereof or ananti-Fc_(ε) R1 antibody or a fragment thereof is used as the firstbinding molecule.
 6. Method as claimed in one of the previous claims,wherein an anti-gp55 antibody or a fragment thereof is used as thesecond binding molecule.
 7. Method as claimed in one of the previousclaims, wherein erythrocytes are lysed or/and leukocytes are fixedbefore step (c).
 8. Method as claimed in claim 7, wherein a wash step iscarried out after fixation.
 9. Method as claimed in one of the previousclaims, wherein the sample is additionally incubated with a thirdbinding molecule which can bind to the surface molecule CD45 in whichthe third binding molecule carries a marker group or can bind to amarker group that can be detected separately in addition to the markergroups of the first and second binding molecule.
 10. Method as claimedin one of the previous claims, wherein directly labelled bindingmolecules are used.
 11. Method as claimed in one of the previous claims,wherein the binding molecules carry fluorescent markers that can bedetected concurrently.
 12. Method as claimed in claim 11, wherein thedetermination is carried out in a flow cytometer.
 13. Method as claimedin one of the previous claims, wherein the sample is additionallyincubated with a DNA dye which can be detected in addition to thebinding molecule labels.
 14. Method as claimed in claim 13, wherein theDNA dye is a fluorescent dye.
 15. Method as claimed in claim 14, whereinthe fluorescent DNA dye is 7-aminoactinomycin D.
 16. Method as claimedin one of the previous claims, wherein the sample is whole blood. 17.Method as claimed in claim 16, wherein the sample is heparinized wholeblood.
 18. Method as claimed in one of the previous claims, wherein thesample is a human sample.
 19. Method as claimed in one of the previousclaims, wherein the incubation of step (a) is carried out for 5-60 minat a temperature of 25-40° C.
 20. Method as claimed in claim 19, whereinthe incubation of step (a) is carried out for 20-40 min at 35-39° C. 21.Method as claimed in one of the previous claims, wherein the incubationof step (b) is carried out for 10-40 min at a temperature of 0-10° C.22. Reagent kit for the determination of the degranulation activity ofbasophilic granulocytes, wherein it contains at least one first bindingmolecule which can bind to cell-bound IgE antibodies and a secondbinding molecule which can bind to a surface molecule which appears denovo on the surface of basophilic granulocytes during degranulation inwhich the first and the second binding molecule carry different markergroups that can be detected concurrently or each can bind to differentmarker groups that can be detected concurrently.
 23. Reagent kit asclaimed in claim 22, wherein it additionally contains a third bindingmolecule which can for example bind to the CD45 surface molecule andcarries a marker group which can be detected in addition to the markersof the first and second binding molecules or can bind to such a markergroup.
 24. Reagent kit as claimed in one of the claims 22 or 23, whereinit additionally contains a DNA dye.
 25. Reagent kit as claimed in one ofthe claims 20 to 24, wherein it additionally contains a reagent forlysing erythrocytes or/and for fixing leukocytes.
 26. Reagent kit asclaimed in one of the claims 22 to 25, wherein the labels arefluorescent labels.
 27. Reagent kit as claimed in one of the claims 22to 26, wherein it contains at least one test substance an anti-gp55 FITCantibody, anti-IgE-PE antibody, FACS lysing solution as well asoptionally 7-aminoactinomycin D or/and an anti-CD45-PerCP antibodyor/and a stimulant.
 28. Reagent kit as claimed in one of the claims 22to 27, wherein it additionally contains conventional buffers, auxiliarysubstances or/and additives.
 29. Method for diagnosing allergichypersensitivity of a patient or the response to a hyposensitization,wherein the degranulation activity of basophilic granulocytes isdetermined in a sample from the patient by means of a method accordingto the invention as claimed in one of the claims 1 to 21 using one orseveral allergens and a positive control as test substances, thedegranulation activity in a negative control sample is determined in anadditional test mixture and the presence or the absence of an allergichypersensitivity or the success or failure of a hyposensitizationtherapy is determined by comparing the values that are obtained.
 30. Useof anti-gp55 antibodies in a method for determining the degranulationactivity of basophilic granulocytes.
 31. Use of a method as claimed inone of the claims 1 to 21 for diagnosing allergic hypersensitivity. 32.Use of a reagent kit as claimed in one of the claims 22 to 28 fordiagnosing allergic hypersensitivity.
 33. Use of a method as claimed inone of the claims 1 to 21 for monitoring the response to ahyposensitization therapy.
 34. Use of a reagent kit as claimed in one ofthe claims 1 to 28 for monitoring the response to a hyposensitizationtherapy.